Application of two-dimensional black phosphorus material in wound healing

2.1 Pathophysiological changes in wound healing

Skin is the first line of defense to protect the body’s internal environment to maintain homeostasis and resist harmful external stimuli [3]. Normal skin tissue has a complex and dense structure. The keratinocytes in the epidermis are constantly self-renewing, replenishing by multiplication and differentiation into the stratum corneum, stratum granulosum, and stratum spinosum, ensuring that the epidermis maintains adequate thickness [23,24]. At the same time, the dense intercellular connections between keratinocytes prevent water and harmful substances from entering the body directly [25]. The dermis, located below the epidermis, contains nerves, blood vessels, lymphatic vessels, and other supporting structures that can provide nutrients to the epidermis [26,27,28]. Meanwhile, the dermis reticulum also contains a large number of collagen fibers and elastic fibers [29]. These interwoven fibers provide toughness and elasticity to the skin and further improve the mechanical strength of the skin to protect tissues and organs in the body.

However, although the skin’s structure is strong, there are limits to its ability to resist outside aggression and damage. When the skin is exposed to strong external factors such as sharp force injury, external force, chemical agents, extreme temperatures, and electric shocks, the integrity of the skin is damaged, and the protective function of the body is lost [30]. When the wound is formed, the body will repair itself through a series of pathophysiological changes. The whole repair process can be divided into four dynamic and continuous stages: the hemostatic stage, inflammatory stage, proliferation stage and remodeling stage (Figure 1) [31,32]. After skin injury occurs, blood cells in damaged capillaries, arterioles, and venules leak into the tissue space, and platelets in plasma are activated under the combined action of endogenous and exogenous coagulation factors, resulting in the formation of platelet thrombus at the site of blood vessel rupture, followed by fibrin [33]. The combined action of fibronectin and blood cells converts into red and mixed thrombi, which form an embolism at the site of injury, typically within 72 h of injury [34]. At the same time, as thrombosis in the hemostatic stage, damaged cells, and formed platelets can release a variety of cytokines and inflammatory factors, such as IL-1β, TNF-α, FGF, and PDGF, into the internal environment together [35,36,37]. These inflammatory factors can promote the migration and aggregation of neutrophils and macrophages to the injured area, and the repair process will then enter the inflammatory stage [38]. In this stage, the neutrophils that first arrive at the injured area release reactive oxygen species (ROS), antimicrobial peptides, proteolytic enzymes and other substances and undergo phagocytosis to remove necrotic cell tissue, foreign substances, and microorganisms in the injured area [39,40]. At the same time, neutrophils can continue to release proinflammatory factors, forming a positive feedback mechanism to promote more neutrophils to converge on the injured area [41]. As necrotic tissue and pathogens are gradually removed, the number of neutrophils is gradually reduced. Most of the remaining neutrophils are squeezed out of the wound area, and a small number of neutrophils are swallowed by the subsequent migration and aggregation of mast cells and macrophages [42,43]. The phenotype of macrophages also gradually transformed from M1, which promoted the release of inflammatory factors, to M2, which promoted angiogenesis and tissue regeneration, and wound repair also entered the proliferative stage [44,45].

Figure 1

The stages of wound repair and their major cellular components [46].

When the repair enters the proliferative stage, keratinocytes, fibroblasts, and endothelial cells begin to accumulate and proliferate under the action of EGF, FGF, VEGF, and other growth factors released by macrophages [47]. Fibroblasts secrete synthetic collagen, proteoglycan, fibronectin, and other protein structures, providing skeletal support for the migration and proliferation of cells in the injured area [48,49]. On this basis, keratinocytes near the injured area migrate to the injured area under the stimulation of growth factors and chemokines and continue to differentiate into mature epidermal cells such as keratinocytes on the basis of proliferation to maintain self-quantity, promoting the re-epithelialization of epidermal tissue [35,50,51]. At the same time, vascular endothelial cells also migrate and proliferate to the wound under the stimulation of VEGF and eventually form a new capillary network to provide nutrition for the new tissue [52,53]. Two or three weeks after injury, wound healing begins to enter the remodeling stage, which is mainly the remodeling of new tissue and the formation of scar tissue. The new granulation tissue is mainly composed of type III collagen with low elastic tension, while the normal skin tissue is mainly composed of type I collagen with higher tensile strength [54]. Therefore, under the action of fibroblasts and collagenase, collagen in granulation tissue is constantly degraded and regenerated to form higher strength type I collagen, thereby improving the mechanical strength of the new skin tissue [55,56,57]. At the same time, the excess capillaries and residual inflammatory cells formed in the repair process will be gradually eliminated by apoptosis, and eventually scar tissue will be formed [58,59].

2.2 Factors affecting wound healing

Normally, the whole wound healing process takes approximately 2–4 weeks from the hemostatic period to the end of the proliferative period. Both endogenous and exogenous factors can affect the process of wound healing, shortening, or prolonging the healing time [60,61]. A younger age brings with it a higher level of metabolic synthesis, thus shortening the time required for wound healing [62]. Vitamin B12, essential fatty acids, zinc, and other essential nutrients and micronutrients can also speed up the healing process [63,64,65]. There are also many factors that affect wound healing and lead to prolonged wound healing time. In addition to the decreased metabolism and longer healing time caused by aging [62,66], chronic diseases such as diabetes and glomerulonephritis can also affect the wound healing process [1]. Prolonged increases in blood glucose concentration affect neutrophil migration to the injured area and microangiogenesis and inhibit keratinocyte and fibroblast-mediated re-epithelialization and extracellular matrix remodeling [67]. Under hyperglycemia conditions, vasodilation function is impaired, and growth factors are subsequently reduced, making it difficult to activate the wound-healing cascade [68]. Furthermore, hyperglycemic stimulation will lead to the accumulation of excessive ROS [69]. Continuous exposure of endogenous proteins, nucleic acids, lipids, and other important biological macromolecules to a high concentration of ROS microenvironment may lead to irreversible destruction of their structure and function, which is not conducive to wound healing [70]. Glomerulonephritis leads to prolonged hypoproteinemia, which affects the synthesis and secretion of cytokines and growth factors and prolongs the entire healing process [71]. The body’s hormone levels also play a role in the healing process. Long-term use of glucocorticoids can inhibit the inflammatory response, slow the aggregation of neutrophils and macrophages to the wound during the inflammatory period, and prolong the wound healing time [72,73]. In addition to the above endogenous factors, nicotine, alcohol, and other exogenous factors related to life habits can also affect the wound healing process. Nicotine and NO in cigarettes can cause small blood vessel contraction, increase platelet adhesion, and cause small blood vessel occlusion; nicotine also causes a decrease in the number of red blood cells and inhibits fibroblast proliferation, resulting in significantly longer wound healing times [74,75]. Nicotine can cause acute damage to the human skin circulatory system by amplifying norepinephrine-mediated skin vasodilation and injure endothelial mediated skin vasodilation [76]. Alcohol inhibits the body’s immune response while reducing the level of collagen-forming metalloproteinases (MMPs), which affects the normal healing of wounds [77].

With the increase in the number and time of exposure to tobacco and alcohol, the degree of skin aging will increase. Studies have found that alcohol affects the lipid composition and cholesterol metabolism of the skin, enhances the permeability of the skin, and thus destroys the barrier function of the skin [78]. In summary, wound healing is a continuous and complex dynamic process affected by multiple factors. Therefore, to achieve rapid and effective wound healing, it is necessary to construct a multifunctional and all-round wound healing platform from multiple angles and levels to achieve rapid wound healing with maximum effect.

2.3 Current demand for tissue engineering materials for wound surfaces

In view of the numerous factors that affect wound healing, for wound tissue engineering, the realization of rapid and efficient wound healing requires that biological materials meet various requirements. First, wound tissue engineering materials need to have good biocompatibility, which is mainly reflected in the fact that they will not cause immune rejection and no cytotoxicity [79]. The bioengineering materials covered on the wound should also have certain mechanical strength and good adhesion, which can be tightly covered on the wound to provide protection [80,81]. On this basis, a good tissue engineering material for the wound should have good absorption capacity and a certain moisture performance, which can not only absorb excessive exudation from the wound but also maintain a certain humidity to avoid adhesion and secondary damage caused by the application of traditional dressings such as gauze [82,83,84]. At the same time, wound tissue engineering materials should also have certain antibacterial and bactericidal abilities, which can maintain a relatively sterile environment of the wound, avoid bacterial and microbial colonization and growth on the wound, and reduce the occurrence of infection [85,86]. Finally, wound tissue engineering materials can also carry some drugs, factors, or stem cells to give the materials the ability to promote cell proliferation and growth and skin regeneration to construct composite wound tissue engineering materials with biological activity [87,88,89].

3.1 Basic structure and properties of BP

BP is a single-element two-dimensional layered material with a lamellar structure similar to graphene, where atomic layers are stacked together through van der Waals force interactions [90]. Within a single layer, each phosphorus atom combines with three adjacent phosphorus atoms in a covalent bond to form a wrinkled honeycomb structure [91]. In addition to forming three covalent bonds, each phosphorus atom retains electron pairs in its outermost orbital (Figure 2). Due to the above unique crystal structure and band structure, BP exhibits high carrier mobility and good optical and mechanical properties and has shown attractive application prospects in gas sensors, solar cells, energy storage devices, and biomedicine [92,93,94].

Figure 2

Structure of BP [101]. (a) The crystal structure of layered BP, (b) the front view of layered BP, (c) the vertical view of layered BP, and (d) the detailed lattice parameter of layered BP.

In the field of biomedical applications, the application of BP, especially BP nanomaterials represented by zero-dimensional BP quantum dots (QDs) and two-dimensional BP, has been increasing [95,96,97]. The unique structure of BP enables BP crystals to be easily stripped into ultrathin nanosheets under external forces [15]. The unique range of the layer-related band gap gives BP a wide range of photon absorption, covering the ultraviolet to NIR region, which makes BP have an excellent photothermal effect and a killing effect on tumor cells [98,99]. In addition, compared with two-dimensional materials such as graphene, the folded structure of BP gives it a larger surface-volume ratio, making it a drug carrier with a very high drug loading rate [100]. More importantly, BP is a low-cost elemental material that can be easily functionalized with other active substances to generate new physicochemical properties and biological properties, further broadening the application prospects of BP in the field of biomedicine.

3.2 Preparation and modification methods of BP

The first preparation of BP occurred over a century ago. In 1914, Bridgman worked at high temperatures of 1.2 GPa and 200℃ [102]. Under pressure conditions, white phosphorus was first converted into BP, and later, under a high pressure of 8.0 GPa, red phosphorus was used as a raw material to obtain BP [103,104]. In addition, it was found that under the presence of shear stress, red phosphorus could also be converted into BP if the pressure was reduced to 4.5. With the constant adjustment of reaction temperature and pressure, various forms of BP crystals, such as needle rods and blocks, have been prepared [105,106]. However, thin layers and single layers of BP applied in biomedicine have received attention and been prepared in large quantities in the last 10 years [103]. Similar to other two-dimensional materials used in biomedicine, the preparation of thin layers of BP can also be divided into top-down and bottom-up types depending on the source of the raw material [107–109]. Here we introduce the representative preparation method of the two kinds of preparation methods. As shown in Table 1, the advantages and disadvantages of different BP preparation methods are summarized.

4.1 Biocompatibility

Good biocompatibility is a basic requirement in applying materials to biomedical engineering. The main component of BP is phosphorus, and its degradation product is mainly phosphate, which can be involved in the regulation of cell metabolism and the homeostasis of the body’s internal environment [147]. Excessive phosphate can also be excreted from the body through the metabolism of the urinary system. Therefore, BP has good biocompatibility and will theoretically not cause serious adverse reactions after implantation in the body [148,149]. Some studies have found that BP nanomaterials can be degraded and excreted over time, and the material can decay to half within 17 h without accumulated toxicity, which indicates that BP nanomaterials have good in vivo degradation [150]. Sun et al. examined the biological distribution of dextran-modified BP materials in BALB/c mice and found that they were mainly present in the liver, spleen, and kidneys [151]. The accumulation of BP in the kidneys was also reported in his study, which found that oral administration of BP resulted in high levels of somatic phosphorus in the kidney tissue, suggesting that at least some BP was distributed and accumulated in the spleen tissue. In practical applications, the cytotoxicity and tissue toxicity of BP are related to the size, concentration, application of photothermal stimulation, and many other factors. Kenry and Lim found that the cytotoxicity of BP was intermediate between that of GO and transition metal disulfide [152]. Zhang et al. found that the larger size and multiple layers of BP nanosheets (transverse size 884.0 ± 102.2 nm, thickness 91.9 ± 32.0 nm) were compared with the smaller size and fewer layers of BP nanosheets (transverse size 208.5 ± 46.9 nm, thickness 17.4 ± 9.1 nm thickness) were more cytotoxic [153]. The safe concentration of BP in organisms is also related to its size. For QDs and mono-layers of BP below 10 nm, even concentrations up to 1,000 μg/mL will not affect cell growth [147]. Furthermore, many studies have shown that the biocompatibility of BP can also be affected by surface modification. It was found that unmodified BP increased neutrophils in peripheral blood of mice, and increased the production of TNF-α, Eotaxin, IL-6, MCP-1, KC, McP-1β, MIG, VEGF, and other pro-inflammatory cytokines, while modified BP significantly eliminated these acute inflammatory responses [154,155]. The size of BP also has an impact on its biocompatibility. With the increase in the size of BP nanosheets, the range of safe concentrations is gradually reduced [147]. However, even so, for the size of most BP nanosheets currently applied in living organisms, the concentration of BP used is still far below the safe concentration threshold, so it can be considered that BP itself has good biocompatibility and can meet the requirements of biomedical engineering applications [33,42]. These results show that BP has low cytotoxicity in vitro and good biocompatibility in vivo, and can be modified to improve its biocompatibility level, reduce or even eliminate the adverse effects on cells, and has the potential for application in the biomedical field.

4.2 Degradability

The phosphorus element in BP is highly reactive with water and oxygen in the air. When BP is placed alone in humid air or water, water absorption and degradation will occur quickly [156,157]. The stability of BP is also significantly affected by light, which can produce different kinds of ROS. ROS reacts with BP to form P _x O _y , which leads to BP degradation. The study found that the ultraviolet part of the spectrum was a major contributor to BP degradation [158,159]. On the one hand, the easy degradation and instability of BP can reduce the cytotoxicity caused by its long-term accumulation in vivo, which is conducive to its application in wound tissue engineering. However, wound repair requires a certain amount of time, and the rapid degradation rate is not conducive to the stable maintenance of its concentration in vivo [160].

In addition, this instability under environmental conditions will destroy the stability of BP, and indirectly reduce material availability and increase costs. In addition, BP nanoparticles are easy to be cleared by mononuclear phagocyte system, resulting in reduced therapeutic efficiency and limiting its clinical application. Therefore, there is an urgent need to find an excellent solution to overcome the shortcomings of BP. It has been found that the BP-matrix material complex constructed by the modification of BP using matrix materials or other biomedical materials can improve its stability and prolong the degradation time while retaining the properties of BP. Huang et al. constructed a silk fibroin-modified BP@SF nanosheet. The BP@SF nanosheet remained stable after 14 days of air exposure compared to the unmodified BP nanosheet and had a final life of 20 days before changing color. However, the unmodified BP nanosheets degraded rapidly, with almost all of them degrading at 14 days (Figure 3) [161]. Zheng et al. prepared an antibacterial hybrid BPNPs@Phy nanosheet loaded with the antibacterial agent emodin (Phy) using a liquid-phase stripping technique. The in vivo stability of this modified hybrid nanosheet was significantly improved compared with that of unmodified BPNPs. The degradation rate of BPNPs exceeded 50% on the third day after implantation, while more than 50% of the modified hybrid BPNPs@Phy nanosheets remained stable until the sixth day [162]. On the other hand, the polymer shell can also minimize the interaction of BP nanomaterials with water and oxygen, thus effectively reducing oxidative degradation. Some studies coated the surface of BP with polydopamine and loaded the drug at the same time, and the experimental results found that this drug delivery system based on BP showed better stability and higher photothermal performance [163]. The modification of BP or its composite with other materials can effectively overcome the defect of too fast degradation rate of BP and improve the stability and service life of the material. At the same time, the above modification method of BP can also give it more functionality, and further enhance its application value.

Figure 3

Stability evaluation under ambient conditions [161]. (a) Photographs of BP@SF and BP@NMP aqueous dispersions after their exposure to air. (b and c) Absorption spectra of BP@SF and BP@NMP aqueous dispersions at different periods of time. (d and e) Raman spectra acquired from BP@SF and BP@NMP aqueous dispersions. (f and g) Photothermal curves of aqueous dispersions of BP@SF and BP@NMP irradiated by an NIR laser.

4.3 Photo-responsiveness

Similar to MXene and other two-dimensional materials, BP has excellent photothermal properties in the NIR band. Sun et al. found that the extinction coefficient of BP, especially BP nanodots at 808 nm, is as high as 14.8 L/g cm, and the photothermal conversion efficiency is 28.4% [164]. In addition to the photothermal effect, BP also has a photodynamic effect, which can produce a large amount of singlet oxygen under NIR light irradiation, with a yield of 9.1% [165]. Such excellent photothermal and photodynamic effects allow BP to exert its function in wound tissue engineering applications in a variety of ways. First, the increase in local temperature under the photothermal effect can directly kill microorganisms that may exist in the wound, and the singlet oxygen stimulated by the photodynamic effect can further kill bacteria by increasing the level of ROS [18,166]. Huang et al. constructed a BPQDs@NH wound dressing encapsulated in a hydrogel for the treatment of diabetic MRSA-infected wounds. The results showed that the local wound temperature rose rapidly to 55℃ under NIR irradiation, the inhibition of MRSA played a synergistic role through ROS production, lipid peroxidation, glutathione, and mechanical destruction, and the total effective sterilization rate was approximately 90% (Figure 4) [167]. Mao et al. constructed a chitosan hydrogel embedded with a thin layer of BP. This hydrogel system can produce extensive and reusable antibacterial effects against gram-negative and gram-positive bacteria by generating singlet oxygen and can maintain bactericidal efficacy against Staphylococcus aureus (94.58%) and Escherichia coli (95.6%) after the fourth bacterial reinfection [22].

Figure 4

The effect of BPQDs@NH with laser irradiation on bacterial killing. (a and b) Bacteriostatic ring photograph and (c–f) the corresponding statistical graphs of inhibition zones of MRSA and Ampr E. coli exposed to different treatments at the same concentrations (a: BPQDs, b: normal saline, c: hydrogel, d: BPQDs@NH). Photograph of the agar plates of (g) MRSA and Ampr E. coli exposed to various treatments at the same concentration of 200 µg/mL. (h and i) The corresponding CFU amounts of MRSA and Ampr E. coli exposed to various treatments.

The good photoresponsiveness of BP can not only produce a killing effect on the microorganisms in the wound but also realize controlled release of drugs and bioactive substances under the temperature regulation of the photothermal effect, providing a good release platform for some therapeutic drugs or healing promoting factors with poor slow-release effects. Zhang et al. constructed a novel controllable response microparticle composed of silk fibroin, gelatin, agarose, and BPQDs and loaded it with growth factors and antimicrobial peptides. Under NIR irradiation, BPQDs can absorb NIR and raise the temperature of the particle to the melting point of gelatin so that the drugs encapsulated in the particle can be gradually released into the environment under the action of reversible phase transition, thus achieving the controlled release of growth factors required for wound healing and promoting wound healing and cardiovascular formation [168]. Li et al. constructed Ag-BP coupled vesicles loaded with silver ions, which could achieve precise controlled release of Ag with the action of NIR in the process of photodynamic therapy and achieve a long-lasting bacteriostatic effect on the wound site (Figure 5) [169].

Figure 5

Illustration of the synthesis and properties of BP Ve-Ag+ [169]. (a) Ag+ can significantly enhance the NIR-II light absorption ability of BP by altering the intrinsic band gap of BP QDs after coupling with BP QDs during the self-assembly process. (b) For the cancer theranostic study, the ROS generated by BP Ve-Ag+ under NIR laser irradiation can induce immunogenic cell death and trigger the disassembly of vesicles to release single BP QD units and Ag+ ions.

4.4 ROS regulation

ROS play an important role in cell growth, migration, and metabolism, and maintaining appropriate ROS levels is necessary for normal physiological function [170]. For wound tissue engineering, the level of ROS in the damaged area dynamically changes during the whole process from wound formation to healing. In the early stage of injury, the death of a large number of cells and the destruction of blood vessels lead to the release of many intracellular components into the local internal environment [171]. These accidental release substances will cause a large amount of ROS, activate the migration and aggregation of neutrophils and macrophages in the internal environment and blood to the injured area, and activate the inflammatory cascade reaction to achieve the removal of the damaged local necrotic tissue [172]. At this stage, ROS mainly play a role in promoting the aggregation of inflammatory cells and activating the inflammatory response, and maintaining a high level of ROS is beneficial for wound healing [173]. At the same time, a high level of ROS can also have a certain killing effect on the microorganisms locally existing in the wound and inhibit the growth of bacteria on the wound [174]. However, with the continuous progress of healing, when entering the regeneration and reconstruction stages, high levels of ROS inhibit the activities of endothelial cells, fibroblasts, and keratinocytes and affect the normal healing of the wound [175,176].

BP, as the most stable allotrope of phosphorus, can eliminate all kinds of active oxygen components. BP has a bidirectional regulatory effect on ROS. On the one hand, the excellent electron transfer ability, abundant edge/defect sites and electrophilic basal surface endowed BP with excellent ROS scavenging activity [158]. ROS are adsorbated on the surface of BP, and unpaired electrons are transferred to the electron-deficient part of the nanomaterials, destroying the structure of ROS [158]. Some studies have found that BP can restore the activity of Nrf2 signaling pathway, increase the expression of heme oxygenase 1 and quinone oxidoreductase, and enhance the antioxidant capacity of cells [177]. Further, in vivo transplantation results also demonstrated that BP-treated NPCs could significantly increase the survival rate and effectively inhibit lipid peroxidation, inflammatory response, and neuronal apoptosis in stroke rats. Hou et al. used BP for ROS-induced acute kidney injury and found that BP can effectively improve the prognosis of acute kidney injury by directly depleting ROS accumulation in kidney tissue [178]. Wang et al. constructed a urokinase-loaded BPN for the treatment of ischemic stroke and showed that BPNs can alleviate ROS-induced ischemia‒reperfusion injury and promote neurological recovery by reducing ROS production [179]. Ding et al. constructed an antioxidant and antibacterial hydrogel coated with 4Oi-BP. In the absence of laser irradiation, BP acted as a carrier to control the release of 4OI and cooperated with it to enhance the antioxidant activity of the dressing, thereby reducing the excessive damage of ROS to endothelial cells, promoting neovascularization, and promoting faster healing of diabetic wounds (Figure 6) [180]. On the other hand, in the presence of light, two-dimensional materials represented by BP can transform molecular oxygen into cytotoxic ROS through the photodynamic effect and improve the level of local oxygen free radicals [181]. The increased concentration of ROS can produce a killing effect on local microorganisms in the wound surface [182]. Liang et al. constructed a dense AgNP-doped BP nanosheet (BPN–AgNP). This doping BPN can significantly increase ROS levels under NIR conditions and has a strong and lasting killing effect on bacteria (Figure 7) [183]. Ran et al. prepared BP@APy-Pt nanoparticles partially modified with Pt nanoparticles and amino-benzyl-2-pyridone (APy) using BP as a photosensitizer. On the basis of improving the biostability of BP, this composite nanoparticle can generate a large amount of singlet oxygen locally through the photodynamic effect and is partially captured under the action of APy for continuous release in the following 24 h, realizing the sustained maintenance of high-level ROS and significantly improving the bactericidal effect on bacteria [184]. By adjusting the local dose, release, and illumination time of BP, bidirectional conversion from removing ROS to releasing ROS can be realized. BP’s bidirectional control ability of ROS levels makes it have excellent application advantages in wound tissue engineering.

Figure 6

4OI–BP@Gel promotes antioxidant activity, alleviates oxidative responsiveness and restores normal MMP in HUVECs [180]. (a) Representative ROS staining of HUVECs under different treatment conditions (scale bar = 50 µm); (b) quantification of fluorescence intensity of (a); (c) the ROS levels of HUVECs in different groups treated or untreated with t-BHP; (d) statistical analysis of ROS levels in HUVECs under different treatment conditions; (e) FACS results of mitochondrial membrane potential in different groups; and (f) statistical analysis of mitochondrial membrane potential.

Figure 7

The antibacterial mechanism of BPN–AgNPs under light irradiation [183]. (a) Photocurrent density of BPNs and BPN–AgNPs. (b) XPS valence spectra of BPNs and BPN–AgNPs. (c) SEM characterization of the morphological features of E. coli treated with BPN–AgNPs under light illumination. (d) Schematic illustration of BPN–AgNPs under light irradiation.

4.5 Electrical conductivity

Bioelectrical activity is an inherent property of human tissue [185]. For skin and soft tissue, when its integrity is compromised, an endogenous electric field is formed in the damaged area, generating a microcurrent at the edge of the wound edge [186]. This biological endogenous electric field stimulates cell migration and growth [187]. It has been proven that endogenous electric fields and exogenous electric fields represented by bioelectric stimulation after injury can stimulate macrophages, lymphocytes, and neutrophils to migrate to the injured area in the early stage of injury, increase blood flow in the injured area, promote tissue fluid return and reduce edema, and stimulate the proliferation and migration of fibroblasts and epithelial cells in the late stage of healing [188,189]. In addition, the wound healing process was accelerated. Therefore, the use of biomaterials to improve the local bioelectrical activity of tissues is very important to accelerate tissue healing. At present, a variety of two-dimensional materials, including GO and MXene, have been applied in the construction of conductive wound tissue engineering materials, and good results have been achieved. Tang et al. constructed a chitosan-fibroin protein scaffold containing GO, which has good electrical conductivity. The experimental results showed that this scaffold combined with electrical stimulation can significantly improve the migration and proliferation of fibroblasts and promote the healing of skin wounds [190]. Zhu et al. constructed an electroactive oxidized alginate/gelatin/MXene composite hydrogel, which has better mechanical properties and electroactivity than alginate/gelatin hydrogel alone and good cytocompatibility with NIH3T3 cells, which can promote fibroblast attachment and migration [191]. The existence of lone electron pairs and high carrier mobility in BP give it good conductivity. Therefore, theoretically, BP can be used as a modified material to improve the conductivity and electrical activity of wound tissue engineering materials, which can also achieve good results. Some studies have found that the electrical conductivity of BP is the best physical cue to regulate stem cell control of neuronal lineage [192]. Xu et al. have demonstrated that the inclusion of dopamine-modified BP in a hydrogel matrix can significantly accelerate the differentiation of bone marrow mesenchymal stem cells into neurons under electrical stimulation [193]. When using BP as a conductive medium, it is necessary to pay attention to how to conveniently and safely carry out electrical stimulation transmission.

Moreover, long-term biosafety should not be overlooked. It has been reported that the toxicity of carbon-based biomaterials depends on their size, dose/concentration, and surface oxygen content, and the mechanism of cytotoxicity may be due to strong interaction with proteins, inducing protein aggregation and leading to the destruction of cell membrane structure [153,194,195]. Therefore, factors such as concentration measurement should be considered when preparing electroactive materials using BP. While ensuring that the biomaterial containing BP has electrical conductivity, it should also ensure that the material has good biocompatibility.

5.1 Improving the tissue repair properties

In skin wound repair, many studies have incorporated BP into other bioactive materials to enhance the tissue repair ability of the materials. Zhou et al. prepared a glucan-based hydrogel composed of gelatin methacrylate and dextran oxide, and used the hydrogel to load BP nanosheets and zinc oxide nanoparticles [197]. The hydrogel showed excellent photothermal properties, broad-spectrum antibacterial activity, and wound healing promotion properties. In the mouse infected wound model, the experimental results showed that the hydrogel could effectively inhibit the wound infection, promote the skin wound healing, significantly increase the new blood vessels in the wound, and shorten the inflammatory response time. In addition to antibacterial and proliferation promotion, hemostasis is also an important feature of the new wound dressing. Wang et al. prepared composite wound repair materials by incorporating BP and copper nanoparticles into chitosan hydrogel to achieve the purpose of hemostasis and promote wound healing [198]. After the addition of BP, the composite showed obvious thermosensitive sponge state, the hemostatic effect of the material was significantly enhanced, and the coagulation index reached 24.98%. In addition to composite hydrogels, composite sponges, etc., composite scaffold systems made of various materials are also used in skin dressing research. Xue et al. developed a nanocomposite scaffold for in situ treatment and wound healing after melanoma surgery by embedding BP into a bioabsorbable gelatin-PCL nanofiber scaffold [199]. The results show that the composite scaffolds undergo sol-gel transformation under NIR stimulation and release BP nanoparticles in situ. In this process, most BP-based nanoagents are selectively internalized by melanoma cells for synergistic photothermal therapy (PTT) and heat-triggered DOX therapy, while some loaded DOX is released into wound tissue, forming a tumor suppressor microenvironment. Furthermore, BP can gradually degrade to phosphates/phosphonates, thereby enhancing tissue repair by activating the ERK1/2 and PI3K/Akt pathways. Considering the anti-melanoma and wound repair effects of the composite scaffold, it may provide a simple strategy for wound treatment after melanoma surgery. By combining BP nanomaterials with various traditional materials, new composite materials such as hydrogel, composite sponge, and composite scaffold were prepared for the preparation of wound dressing. These dressings not only show the inherent properties of BP, but also play the functional properties of other traditional materials, which can cooperate and complement to increase the tissue repair ability of dressings, achieve the purpose of promoting wound healing, provide a new method for wound treatment, and provide a new idea for the research and development of new wound dressings.

5.2 Mxene matrix material-assisted PTT

PTT uses photothermic agents as internal energy absorbers to locally convert NIR light energy into heat energy, generating high heat and causing target cell necrosis or apoptosis. More and more scholars have found that PTT can promote skin wound healing in addition to treating cancer [200].

Because of its good optical properties, BP is also widely used in the treatment of skin injuries with PTT. Composite nanosheets have been formed by loading antibacterial agents onto the surface of BP via hydrophobic interactions [162]. The results showed that the composite nanosheets had good stability and low cytotoxicity under physiological conditions. In addition, the synthesized composite nanosheets have excellent photothermal conversion ability. After irradiation of 808 nm NIR light, light energy is converted into heat energy, promoting antibacterial agents physical release. Under the synergistic effect of PTT and antibacterial agents, the composite nanosheets have excellent bactericidal effect against Staphylococcus aureus (99.7%) and Pseudomonas aeruginosa (99.9%). Ding et al. incorporated 4OI-modified BP nanosheets into the photosensitive gelatin methylacrylamide hydrogel to prepare a novel photothermal and photodynamic treatment system with antibacterial and antioxidant properties for diabetic wound regeneration [180]. Under laser irradiation, hydrogels containing 4OI-modified BP nanosheets can gel quickly, form a film on the wound, which can eliminate bacterial infections.

As a carrier, BP controls the release of 4OI and collaborates with 4OI to enhance antioxidant activity, thereby reducing the damage of excessive ROS to endothelial cells, promoting neovascularization and promoting faster healing of diabetic wounds. The above research results make full use of the photothermal properties of BP nanomaterials, and the photothermal agent is used as a composite material to produce antibacterial properties, and synergies with the antibacterial properties of its own or other materials, making BP one of the best alternative antibiotics to solve drug-resistant and infected wounds.

5.3 MXene matrix material-assisted electrical stimulation therapy

When the epithelium is damaged, it generates its own endogenous direct current electric field (DCEF) and transepithelial potential differences, which drive current out of the damaged area and continue locally until the healing process is complete [201]. The physiological DCEF generated by epithelial injury can actively regulate cell behavior, promote angiogenesis, block edema formation, down-regulate inflammatory factors, promote granulation tissue formation, and promote collagen synthesis [186]. Some studies have found that wound healing is slowed when the electric field is removed, suggesting that electrical stimulation can be effective in promoting skin wound healing; however, most wound dressings are not electrically active and therefore do not have any effect on the wound site during healing [186,202]. Therefore, an ideal epidermal wound dressing, in addition to providing a physical platform for cell growth and tissue repair, should also allow local current transfer to the wound site. Liu et al. added different amounts of BP into polymer materials, and the experimental results found that electric stimulation alone, polymer materials and polymer materials containing BP did not harm the survival of normal cells, and ES could also promote cell proliferation and differentiation [203]. The ability of electrical stimulation combined with BP in promoting tissue healing was verified by the observation of wound morphology, wound healing rate, and histomorphology of rabbit during treatment. In summary, biodegradable electroactive BP material is a promising candidate material for skin wound healing dressing. Using the good electrical conductivity of BP combined with electrical stimulation is an effective synergistic treatment strategy, which is of great significance for the development of auxiliary electrical stimulation for wound treatment.